Mast cells are key immune effectors cells in IgE-dependent responses in allergic disorders and parasite infections. Mast cells also play an important role in innate immunity against a variety of gram-negative bacteria and development of autoimmune disorders. In order to develop new treatment strategies for these mast cell-related disorders, it is critical to understand the mechanisms of mast cell development. In the past several years, we have purified myeloid or lymphoid hematopoietic progenitors, which have been very useful to analyze molecular events involved at each checkpoint in normal hematopoiesis. In this study, we will try to delineate the mast cell developmental pathway from other myeloid pathways, and will evaluate their developmental checkpoints. We recently found that mast cell progenitors (MCPs) can originate from granulocyte/monocyte progenitors (GMPs) sharing the origin with neutrophils and monocytes. Furthermore, we have successfully isolate progenitors bipotent for mast cells and basophils in the spleen (basophil/mast cell progenitor: BMCP) by using a multi-color FACS system. By culturing GMPs, we isolated different stages of mast cell-committed progenitors (MCPs). By using these purified MCPs, we will evaluate the role of transcription factors essential for mast cell development such as PU.1, GATA-2 and MITF, and of neutrophils/monocyte-related transcription factor, C/EBPalpha, in mast cell development. In steady-state hematopoiesis, the intestine collects the vast majority of mast cell colony-forming activity, and therefore the intestine could function as a reservoir of mast cells for tissues outside the intestine. We will identify intestinal MCPs by utilizing the method used to identify BMCPs and GMP-derived MCPs, and will characterize their biological activity and developmental mechanisms. We will then try to identify bone marrow cell population that seeds the intestine to give rise to intestinal MCPs. Results obtained from this project will be critical to study mast cell biology, which eventually will help develop new therapeutic strategies for a variety of allergic and autoimmune disorders.